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Experimental coccidiosis provoked by Eimeria acervulina in chicks simultaneously fed on ochratoxin A contaminated diet
Research in Veterinary Science 82 (2007) 225–231 www.elsevier.com/locate/rvsc
Experimental coccidiosis provoked by Eimeria acervulina in chicks simultaneously fed on ochratoxin A contaminated diet V. Koynarski a, S. Stoev
b
b,*
, N. Grozeva b, T. Mirtcheva c, H. Daskalov d, J. Mitev e, P. Mantle f
a Department of Parasitology, Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria Department of General and Clinical pathology, Faculty of Veterinary Medicine, Trakia University, Stara Zagora 6000, Bulgaria c Department of Biochemistry, Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria d Department of Veterinary Hygiene, Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria e Department of Ecology and Zoochygiene, Faculty of Agriculture, Trakia University, Stara Zagora, Bulgaria f Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK
Accepted 5 July 2006
Abstract The progression of coccidiosis provoked by Eimeria acervulina was followed in chicks fed on OTA-contaminated as well as on OTAfree diets. More heavy progress of duodenal coccidiosis, including mortality, occurred in OTA-treated chicks as can be seen from the higher value of lesion (3.50) and oocyst (31.65) indices. A stronger decrease of serum total protein was found in OTA-treated chicks (22.80 g/l) than in chicks infected with E. acervulina(24.20 g/l), but that decrease was strongest in chicks treated with OTA and simultaneously infected with E. acervulina (19.71 g/l). The serum concentration of uric acid was significantly increased in all chicks exposed to OTA, most notably in those additionally infected with E. acervulina (1020.6 (l mol/L), whereas the serum enzyme activity of AST was increased only in chicks infected with E. acervulina and highest in those fed OTA contaminated diet (122.2 U/L). OTA induced degenerative changes in kidneys, liver and heart as well as a depletion of lymphoid tissue in the lymphoid organs and a decrease of body weight. Coccidiosis induced only a slight growth depression and duodenal hemorrhages in addition to characteristic duodenal damages. The impairment of kidney function, histopathological changes and general growth depression were stronger when chicks infected with E. acervulina were also given OTA. 2006 Elsevier Ltd. All rights reserved. Keywords: Ochratoxicosis; Ochratoxin A; Coccidiosis; E. acervulina; Pathology
1. Introduction Ochratoxin A (OTA) is a widespread mycotoxin in lots of components of animal feed. Therefore, the significance of this mycotoxin for animal health has been comprehensively reviewed (Kuiper-Goodman and Scot, 1989; Marquardt and Frolich, 1992). The most important economic problems reported when OTA-contaminated feed was given in chicks were the growth depression and increased mortality, although some other specific damages on many *
Corresponding author. Fax: +359 42 670624. E-mail address: [email protected] (S. Stoev).
0034-5288/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2006.07.004
tissues were also noted (Huff et al., 1974; Dwivedi and Burns, 1984, 1986; Stoev et al., 2000a, 2002b,c, 2004). The increased mortality among chicks or pigs fed on OTA-contaminated diet was seen to be due to OTA-provoked susceptibility to secondary bacterial infections (Stoev et al., 2000b) or to a heavy progression of some often encountered parasitic diseases (Stoev et al., 2002b), because of the suppression in both humoral and cell-mediated immune response in such animals as an aspect of ochratoxicosis (Burns and Dwivedi, 1986; Stoev et al., 2000a,b, 2002c). Our observations on naturally encountered chick nephropathy in Bulgaria have identified a rather high incidence of mortality which can only partly be attributed to
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OTA-contaminated feed (Stoev et al., 2002a). The descriptions of pathology of spontaneous chick nephropathy vary considerably, especially if another secondary disease arises spontaneously along with OTA-induced nephropathy in chicks (Stoev et al., 2002b) that can occur frequently because of the immunosuppression accompanying this nephropathy (Stoev et al., 2000a,b). Therefore, it is important to clarify the complex clinicomorphological findings in such cases, which derive from both diseases as well as to follow the progression of coccidiosis in the same cases. At present, there are very scarce and contradictious data on progression of coccidiosis in chicks fed on OTA-contaminated diet. For example, Huff and Ruff (1982) reported an increase of serum uric acid and an increase of relative weight of liver in chicks fed OTA and infected with E. tenella, which was confirmed by Stoev et al. (2002b), but both the relative weight of liver and the uric acid concentration in serum were decreased in chicks fed OTA and infected with E. acervulina (Huff and Ruff, 1982). That’s why, we decided to extend our recent study on progression of coccidiosis provoked by E. tenella in chicks fed on OTAcontaminated diet (Stoev et al., 2002b) and included E. acervulina in our further investigations in this area.
Table 1 Experimental design Group
OTA in feed between days 1 and 14 (ppm – mg/kg)
Inoculation with E. acervulina – day 7 (4 · 105 oocysts per chick per os)
I II III
None 4 4
E. acervulina None E. acervulina
Control
None
None
Feed levels of ochratoxin A (given between days 1 and 14 of the experiment) and inoculation with E. acervulina (at day 7 of the experiment) of various experimental groups of chicks.
2.3. Measurements The chicks were weighed at the beginning of the experiment and after one and two weeks. For following the pathomorphological changes in various internal organs as well as oocyst and lesion indices the chicks from each group were slaughtered two weeks after the beginning of the experiment or one week after the infection with E. acervulina, as the first mortalities occurred in group III in that time (Table 1).
2. Materials and methods
2.4. Histological examination
2.1. OTA production
Tissues for histological examination were taken from kidneys, liver, lung, heart, thymus, bursa of Fabricius, spleen, intestine, cerebellum, brain and medulla and fixed in 10% neutral buffered formalin. The fixed tissues were processed for paraffin embedding, sectioned at 6 lm and stained with haematoxylin–eosin.
Aspergillus ochraceus (isolate D2306, as used by Tapia and Seawright, 1984; and Stoev et al., 2000b, 2002b) was grown on sterilized shredded wheat (40 g) in 500 ml conical flasks, moistened by a 40% (w/v) addition of sterile water and incubated on a rotary shaker at 27 C for 2 weeks (Harris and Mantle, 2001). The brown granular product, which bore no obvious sign of fungal growth or sporulation, was sterilized at 80 C for 1 h and stored at 20 C. A sample was analyzed for ochratoxins; batches typically contained OTA (about 2 mg/g) and relatively small component of the biologically-inactive alkaloid ochratoxin B (deschloro-ochratoxin A). No other mycotoxins were produced in this solid substrate fermentation process and the necessary dilution by approximately 103 when homogenized into chick ration made only a minimal addition of other components of the molded shredded wheat substrate (Stoev et al., 2000b, 2002b).
2.5. Parasitological examination The required quantity of oocysts was obtained from naturally susceptible chicks (two-weeks of age) inoculated experimentally with oocysts of E. acervulina according to Lozanov (1980). The oocysts were preserved in 2.5% solution of potassium bichromate (K2Cr4O7) at 4 C until used for inoculation (Table 1). The oocyst index in slaughtered chicks was examined according to Cucler (1959), whereas the lesion index in the same chicks was evaluated according to Johnson and Reid (1970) (Table 2). 2.6. Hematological and biochemical examination
2.2. Experimental design Specific pathogen-free chicks (Plymouth Rock) were purchased at a week of age and housed in wire floor cages with continuous infra-red lighting at a temperature suitable for their age. Commercial complete standard feed without any medication (Smesler, Stara Zagora, Bulgaria) with or without added OTA was available ad libitum. The chicks were housed in 3 experimental and 1 control groups (12 birds in each) and treated either or both with OTA and E. acervulina (Table 1).
Blood samples were taken at slaughter at the end of the 14th day of the experiment and examined for various biochemical parameters within 1–2 h of collection, immediately after separation of serum. The serum total protein was measured by Bio-La-Test (Lachema Diagnostica, Brno, Czech Republic) and uric acid by EnzUric-FT-test (Labordiagnostica, Gopecke, Germany). The serum enzyme activity of AST (asparate-aminotransferase) was measured by Cormay test (Smolenskiego, Warsaw, Poland).
V. Koynarski et al. / Research in Veterinary Science 82 (2007) 225–231 Table 2 Mean values of lesion and oocyst indices in slaughtered chicks (7 days after inoculation with E. acervulina). Group
Number of examined chicks
Lesion indexb
Oocyst indexc
I (E.ac.) II (OTA) III (OTA + E.ac.)
12 12 8
2.41 ± 0.31a 0 3.50 ± 0.26
26.31 ± 0.96a 0 31.65 ± 0.52
Control
12
0
0
± SEM (standard error of the mean). a Significant difference compared to group III (p < 0.05). b Lesion index (the maximum is 4) was examined using the following scheme: assessment 1 – scattered white plaque-like lesions confined to the duodenal mucosa (maximum 5 lesions per square centimeter); assessment 2 – lesions are much closer together, but not coalescent; the intestinal wall shows no thickening; assessment 3 – lesions are numerous enough to cause coalescence in the lesion size, giving the duodenal mucosa a coated appearance; the intestinal wall is thickened and the content is watery ; assessment 4 – coalescing of the lesions is much more complete and no distinctive lesions may appear on the duodenal mucosa; the intestinal wall is considerably thickened. c Oocyst index (the maximum is 40) was examined using the following scheme: assessment 0 – from 0 up to 0.1 million oocysts in the intestine for chick; assessment 1 – from 0.1 up to 1 million oocysts; assessment 10 – from 1 up to 5 millions oocysts; assessment 20 – from 5 up to 10 millions oocysts; assessment 40 – more than 10 millions oocysts.
2.7. Statistical methods Non-parametric Mann–Whitney in addition to Student’s t-test was used to estimate significant differences between the mean values of various parameters in different groups of chicks. 3. Results 3.1. Clinical observation Diarrhoea was the main clinical sign seen first in chicks given OTA (groups II and III) on day 3 from the beginning of the experiment, but the same was also seen in chicks of group I, 5 or 6 days after inoculation with E. acervulina and was most pronounced in chicks infected with E. acervulina and simultaneously given OTA (group III) at that time. Other clinical signs, depression, weakness and dullness, ruffled feathers, reduction of feed intake and growth depression occurred in all experimental groups though mainly in groups II and III. One chick from group III dead at
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day 13 and three more dead at day 14 of the experiment as was expected from the higher values of lesion and oocyst indices (Table 2) and the significant decrease of body weight (Table 3) in slaughtered chicks of the same OTAtreated group. 3.2. Body weight A statistically significant decrease of body weight was observed in all experimental groups compared to the control group, but was most marked in groups given OTA. A significant decrease of body weight was seen in chicks given OTA and simultaneously infected with E. acervulina compared to chicks given only OTA (Table 3). 3.3. Biochemical and hematological findings Total protein in serum was significantly decreased in chicks of all experimental groups, most notably in those treated with OTA and especially in group treated with OTA and E. acervulina simultaneously (Table 4). A significant increase was observed in serum uric acid in all OTA-treated groups, but was highest in chicks additionally infected with E. acervulina (Table 4). Serum enzyme activity of AST was somewhat increased in all experimental groups, but that increase was confident only in groups infected with E. acervulina (Table 4). 3.4. Gross pathology and parasitological findings There were a few subcutaneous muscular hemorrhages in chicks of OTA-treated groups II and III. The kidneys and liver in these chicks were congested and enlarged. Their gall bladder was also distended by bile. The mucosal surface of small intestine was hyperemic and covered with mucous fluid. The pancreas was pink-coloured. The meninges were slightly hyperemic. In chicks infected with E. acervulina (groups I and III) there were various white plaque-like lesions confined to the duodenal mucosa, which sometimes coalesced, most notably in OTA-treated chicks, giving the intestinal mucosa a coated appearance. Also, the mucosal surface of duodenum was hyperemic and coated with mucous fluid. In addition, some red spots (2–3 mm diameter) were seen under duodenal serosa. The intestinal wall of the duode-
Table 3 Mean values of body weight (b.w.) in chicks of various experimental groups at the beginning and at end of the first and second weeks of the experiment Group
Day 1 b.w. (g)
Day 7 Number of chicks
b.w. (g) a
Day 14 Number of chicks
b.w. (g) a,*
Number of chicks
I (E.ac.) II (OTA) III (OTA + E.ac.)
85 ± 4.0 83 ± 3.5 86 ± 2.8
12 12 12
152.9 ± 7.7 103.1* ± 5.5 95.4* ± 6.0
12 12 12
243.7 ± 11.3 155.4a,* ± 12.7 110.0* ± 9.5
12 12 8
Control
84 ± 3.6
12
160.4a ± 6.6
12
314.6a ± 16.8
12
± SEM (standard error of the mean). a Significant difference compared to group III (p < 0.05). * Significant difference compared to controls (p < 0.05).
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Table 4 Mean serum values of total protein, AST (asparate-aminotransferase) and uric acid in chicks of various experimental groups at day 14 of the experiment. Group
Total protein (g/l) a,*
AST (U/L) *
Uric acid (lmol/L) a
Number of chicks
I (E.ac.) II (OTA) III (OTA + E.ac.)
24.20 ± 0.89 22.80* ± 1.74 19.71* ± 0.81
114.8 ± 4.7 99.6a ± 4.8 122.2* ± 7.1
165.5 ± 32.6 872.6* ± 96.7 1020.6* ± 212.4
12 12 8
Control
35.40a ± 0.87
79.0a ± 8.6
214.6a ± 20.9
12
± SEM (standard error of the mean). a Significant difference compared to group III (p < 0.05). * Significant difference compared to controls (p < 0.05).
num was often thickened and the content was watery mainly in OTA-treated chicks as can be seen from the lesion indices (Table 2). The number of oocysts in the intestine and the values of the oocyst indices were also clearly higher in chicks given OTA in diet (Table 2). 3.5. Histopathology No histopathological changes were found in internal organs of control chicks. In kidneys of OTA-treated groups (II and III) peritubular capillaries were congested. Degenerative changes (granular degeneration or cloudy swelling as well as karyopycnosis or karyolysis) in epithelial cells of the proximal convoluted tubules were observed (Fig. 1) as well as focal mononuclear cells infiltration was seen in the renal interstice. Some of the tubules contained necrotic debris, granular or hyaline casts in the lumen (Fig. 2). A limited proliferation of connective tissue and activation of capillary endothelium were also noticed in the same chicks. These damages were stronger in chicks given OTA and E. acervulina (group III) than in those given OTA alone (group II). In chicks treated with E. acervulina alone (group I) only a slight hyperemia of peritubular capillaries and scarce granular degeneration in epithelial cells of the proximal convoluted tubules were seen sometimes. Cloudy swelling, granular or vacuolar degeneration were seen in the livers of OTA-treated groups, but the
same were more marked in group III, infected also by E. acervulina(Fig. 3). Also, there was an activation of capillary endothelium and Kupffer’s cells, hyperemia and pericapillary edema, in addition to perivascular infiltration of mononuclear cells. Only a slight hyperemia of capillaries and scarce granular degeneration in hepatocytes were occasionally seen in chicks of group I, treated with E. acervulina alone. Degenerative changes in lymphoid organs were only seen in chicks of groups II and III. There was a pronounced depletion of lymphoid cells in the cortical zone
Fig. 1. Photomicrograph of kidney in chick fed on 4 ppm OTA in diet and infected with E. acervulina. Granular degeneration and karyopycnosis in epithelial cells of the proximal tubules. H/E, scale bar = 33.3 lm.
Fig. 3. Photomicrograph of liver in chick fed on 4 ppm OTA in diet and infected with E. acervulina. Granular and vacuolar degeneration, karyopycnosis and karyolysis in hepatocites H/E, scale bar = 33.3 lm.
Fig. 2. Photomicrograph of kidney in chick fed on 4 ppm OTA in diet. Necrotic debris and hyaline drops in the lumen of some tubules. H/E, scale bar = 33.3 lm.
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of the thymus and sometimes the region between medulla and cortex was not well defined or the cortex became very thin. Similar depletion of lymphoid cells (Fig. 4) and degenerative changes (karyopyknosis and karyorrhexis) in the lymph follicles were seen in the bursa of Fabricius. A slight cellular depletion or degenerative changes were also observed in the white pulp of germinal centres in the spleen of chicks in the same groups. No histopathological changes were seen in the lymphoid organs in chicks of group I, excepting the slight hyperplasia of the white pulp of the spleen. Degenerative and slight necrotic changes as well as a desquamation of surface- or glandular mucosal epithelium of duodenum and jejunum were seen in chicks of groups II (Fig. 5) and III, but the degenerative changes in the duodenum were stronger in the chicks of group III. Small petechial hemorrhages, hyperemia of vessels, slight mononuclear infiltration in the lamina propria, lots of oocysts and third generation of schizonts, in addition to local degenerative changes of surface- or glandular epithelium were also seen in the mucosa of duodenum in chicks
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Fig. 6. Photomicrograph of duodenal mucosa in chick fed on 4 ppm OTA in diet and infected with E. acervulina. Presence of many preliminary forms of oocysts (schizonts) in intestinal mucosa. Damaged distal part of the villi. H/E, scale bar = 50 lm.
Fig. 7. Photomicrograph of brain in chick fed on 4 ppm OTA in diet and infected with E. acervulina.A pericellular and pericapillary edema. H/E, scale bar = 50 lm. Fig. 4. Photomicrograph of bursa of Fabricius in chick fed on 4 ppm OTA in diet and infected with E. acervulina. Depletion of lymphoid cells in the central part of the lymph follicles. H/E, scale bar = 50 lm.
Fig. 5. Photomicrograph of duodenal mucosa in chick fed on 4 ppm OTA in diet. Degenerative changes and desquamation of glandular- and surface mucosal epithelium, including totally damaged villi. H/E, scale bar = 50 lm.
infected with E. acervulina (groups I and III), most notably in group III (Fig. 6). The distal part of the villi of duodenal mucosa was often with desquamated epithelium or totally damaged in the chicks of the same groups, most notably in group III. A perivascular edema, lytic changes and irregular staining, as a result of the increased eosinophilia of some myofibrils were seen in the heart of chicks of groups II and III. Slight granular degeneration of myofibrils and activation of capillary endothelium were also observed in these chicks. In chicks treated with E. acervulina alone (group I) only a hyperemia of vessels, slight perivascular mononuclear infiltration or scarce granular degeneration of myofibrils were rarely seen. In the lung, there was only a perivascular or peribronchial mononuclear cell infiltration and slight edematous changes in the chicks of OTA-treated groups. In the brain, a pericellular or pericapillary edema was seen in chicks of groups II and III (Fig. 7). In neurons and glia cells of the brain in the same chicks there were
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Fig. 8. Photomicrograph of cerebellum in chick fed on 4 ppm OTA in diet and infected with E. acervulina. Edema in the region of the Purkinje’s cells and slightly in the granular layer. Lytic and pycnotic changes in Purkinje’s cells. H/E, scale bar = 50 lm.
slight lytic changes. In the cerebellum of these chicks, edema and slight degenerative changes were observed in the region of the Purkinje’s cells and rarely in molecular or granular layers (Fig. 8). Slight edematous changes in the white substance of the cerebellum were occasionally seen in the chicks of groups II and III. In the lumbosacral region of the medulla, slight lytic changes in neurons were also noticed in some chicks of the same groups. Only a slight hyperemia of capillaries and scarce lytic changes in neurons were occasionally seen in chicks of group I, treated with E. acervulina alone. 4. Discussion It was found that intensity of clinical signs, macroscopic and histopathological changes, the growth depression as well as impairment of kidney function, expressed by serum levels of uric acid, were stronger when chicks infected with E. acervulina were also treated with OTA. Moreover, the coccidiosis progresses in a heavy way and rapidly in OTA-treated chicks, as perceived from the changes in lesion and oocyst indices and especially from the chick mortality. That could be explained by OTA-provoked immunosuppression and following sensitivity to various infectious and parasitic diseases (Stoev et al., 2000a,b, 2002b). The growth depression in OTA-treated chicks, according to some authors (Mohiudin et al., 1993), may be due to the impairment of protein synthesis by OTA. The histopathological changes in OTA-treated chicks, especially those found in kidneys, corresponded well to the deviation of some biochemical parameters, as the increase in the serum concentration of uric acid, which showed impairment in kidney function was evident. The degenerative changes in the epithelial cells of kidneys and liver probably are due to the route of elimination of OTA via kidneys and partly via liver, due to enterohepatic
recirculation and hepatobiliary way of excretion of OTA (Fuchs, 1988), exerting direct toxic effect of OTA on these organs (Dwivedi and Burns, 1984; Stoev et al., 2000a). The slight granular degeneration in hepatocytes in chicks treated with E. acervulina alone could be due to the resorption of toxic products as a consequence of decomposition of necrotic epithelial debris in the intestinal content (Lozanov, 1983). The degenerative changes and the depletion of lymphoid cells in the lymphoid organs in OTA-treated chicks can be in good agreement with immunosuppressive effects that OTA can exerts in contamination levels 2–4 ppm, as reported by some authors (Holmberg et al., 1988; Harvey et al., 1992; Kozaczynski, 1994). Therefore, a heavy progression of some infectious (Stoev et al., 2000a,b) and even parasitic diseases (Stoev et al., 2002b) could be expected as was demonstrated in the present experiment. The OTAprovoked immunosuppression could be explained by the inhibition of protein synthesis and the subsequent delay in cell division in the immune system (Harvey et al., 1992). The impaired protein synthesis in lymphocytes could lead to the impairment in their activation, differentiation and proliferation. In addition, the decreased phagocytic activity of natural killer cells and T-killer cells, which is probably due to a decrease of basal interferon (Harvey et al., 1992), may additionally deteriorate the immune response of OTA-treated chicks. By the present experiment, it was demonstrated in practice, how the known OTA suppression of humoral and cellular immunity (NNT, 1991; Stoev et al., 2000a,b) can make worse the progression of coccidiosis provoked by E. acervulina, and can also lead to mortality among chicks at day 7 of the infection that cannot be commonly provoked by E. acervulina (Williams, 1973; Lozanov, 1983; Holdworth et al., 2004). Diarrhoea and growth depression in chicks were often reported as the main clinical signs in coccidiosis provoked by E. acervulina, but no mortality was seen in such chicks (Lozanov, 1983). The edematous changes seen in various internal organs, in addition to some muscular hemorrhages, might be due to vascular damages, provoked by OTA (Stoev et al., 2000a). Furthermore, a disturbance of blood clotting due to a reduction in the concentration of fibrinogen in the blood and an increase in the prothrombin time, observed as a response of ochratoxicosis (Doerr et al., 1981; Prior and Sisodia, 1978), could also contribute to these muscular hemorrhages. However, the scarce petechial hemorrhages in the duodenum are commonly observed, because of localization of E. acervulina in this place (Johnson and Reid, 1970). It can be supposed that the significant decrease in the serum total protein could be due to the impairment of protein synthesis, provoked by OTA, as has been previously reported (Mohiudin et al., 1993; Stoev et al., 1999, 2000a, 2002b), but the slight decrease in the serum total protein in the chicks infected only with E. acervulina is likely a consequence of progression of coccidiosis and can explain the
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most pronounced decrease in this parameter in the chicks treated with E. acervulina and OTA simultaneously. The strong increase in the serum level of uric acid in both groups treated with OTA showed that the function of kidneys was significantly impaired, which is in agreement with our previous investigations on OTA-toxicity (Stoev et al., 2000a, 2002b). However, the results of the present study contradict with the conclusion made by Huff and Ruff (1982) that the serum concentration of uric acid decreased in chicks fed OTA and simultaneously infected with E. acervulina as was reported in the same paper. Concerning the serum enzyme activity of AST, it can be concluded that the strongest increase in this parameter in the chicks fed OTA and simultaneously infected with E. acervulina may be due to the strongest degenerative changes in the internal organs of the same chicks. Finally, the present experiment was demonstrated in practice that some often encountered parasitic diseases, as coccidiosis provoked by E. acervulina, can progress in a more heavy way in OTA-treated chicks, and can also lead to mortality among chicks that cannot be commonly provoked by E. acervulina. Such mortality was only characteristic for coccidiosis provoked by E. tenella,but even in such cases coccidiosis developed more rapidly and heavily in OTA-treated chicks as was seen from our previous experiment (Stoev et al., 2002b). However, our observation is in contrast with the study made by Huff and Ruff (1982), which found less lesions in chicks challenged with either E. acervulina or E. tenella and fed OTA compared to chicks not fed OTA. The clinicomorphological finding in such cases may be complicated, because it derives from both diseases. Acknowledgement This study was financially supported by the foundation of Ministry of science and education in Bulgaria. References Burns, R.B., Dwivedi, P., 1986. The natural occurrence of ochratoxin A and its effects in poultry. A review. Part 2. Pathology and immunology. World’s Poultry Science Journal 42, 48–55. Cucler, A.C., 1959. The Laboratory Evaluation of Coccidiostatic Drugs. In: Conference on Methods of Testing Coccidiostats. Merc Chemical Division, Rahway, NJ, Sec. 2, 1–14. Doerr, J.A., Huff, W.E., Hamilton, B.P., Lillehoj, E.B., 1981. Severe coagulopathy in young chickens produced by ochratoxin A. Toxicology and Applied Pharmacology 59, 157–163. Dwivedi, P., Burns, R.B., 1984. Pathology of ochratoxicosis A in young broiler chicks. Research in Veterinary Science 36, 92–103. Dwivedi, P., Burns, R.B., 1986. The natural occurrence of ochratoxin A and its effects in poultry. A review. Part 1. Epidemiology and toxicity. World’s Poultry Science Journal 42, 32–47. Fuchs, R., 1988. Distribution and fate of ochratoxin A in experimental animals. Doctoral thesis, Uppsala, pp. 13–41. Harris, J.P., Mantle, P.G., 2001. Biosynthesis of ochratoxins by Aspergillus ochraceus. Phytochemistry 58, 709–716. Harvey, R.B., Elissalde, M.H., Kubena, L., Weaver, E.A., Corrier, D.E., Clement, B.A., 1992. Immunotoxicity of ochratoxin A to growing gilts. American Journal of Veterinary Research 53, 1966–1970.
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Holdworth, P.A., Conway, D.P., McKenzie, M.E., Dayton, A.D., Chapman, H.D., Mathis, G.F., Scinner, J.T., Mundt, H.C., Williams, R.B., 2004. World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines for evaluating the efficacy of anticoccidial drugs in chickens and turkeys. Veterinary Parasitology 121, 189–212. Holmberg, T., Thuvander, A., Hult, K., 1988. Ochratoxin A as a suppressor of mitogen induced blastogenesis of porcine blood lymphocytes. Acta Veterinaria Scandinavica 29, 219–223. Huff, W.E., Ruff, M.D., 1982. Eimeria acervulina and Eimeria tenella infections in ochratoxin A-compromised broiler chickens. Poultry Science 61, 685–692. Huff, W.E., Wyatt, R.D., Tucker, T.L., Hamilton, P.B., 1974. Ochratoxicosis in the broiler chickens. Poultry Science 53, 1585–1591. Johnson, J., Reid, W.M., 1970. Anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chickens. Experimental Parasitology 28, 30–36. Kozaczynski, W., 1994. Experimental ochratoxicosis A in chickens. Immunological study. Bulletin of Veterinary Institute of Pulawy 38, 1–8. Kuiper-Goodman, T., Scot, P.M., 1989. Risk Assessment of the Mycotoxin Ochratoxin A. Biomedical and Environmental Sciences 2, 179– 248. Lozanov, L., 1980. Studies on the clinic and pathomorphology of some Eimeria invasions in chicks with a view to differential diagnosis. Doctoral thesis, Stara Zagora, pp. 3–31 (Bulg.). Lozanov, L., 1983. On the clinic, morphology and hisokinesis of birds experimentally infected with Eimeria acervulina. Veterinary Science 20, 64–71 (Bulg.). Marquardt, R.R., Frolich, A.A., 1992. A review of recent advances in understanding ochratoxicosis. Journal of Animal Science 70, 3968–3988. Mohiudin, S.M., Warasi, S.M.A., Reddy, M.V., 1993. Haematological and biochemical changes in broiler chicken. Indian Veterinary Journal 70, 613–617. NNT (The Nordic Working Group on Food Toxicology and Risk Evaluation), 1991. Health Evaluation of ochratoxin A in Food Products. Nordiske Seminar-og Arbejdsrapporter (1991 : 545) pp. 5–26. Prior, M.C., Sisodia, C.S., 1978. Ochratoxicosis in White Leghorn hens. Poultry Science 57, 619–623. Stoev, S.D., Anguelov, G., Pavlov, D., Pirovski, L., 1999. Some antidotes and paraclinical investigations in experimental intoxication with ochratoxin A and penicillic acid in chicks. Veterinarski arhiv 69, 179–189. Stoev, S.D., Anguelov, G., Ivanov, I., Pavlov, D., 2000a. Influence of ochratoxin A and an extract of artichoke on the vaccinal immunity and health in broiler chicks. Experimental and Toxicologic Pathology 52, 43–55. Stoev, S.D., Goundasheva, D., Mirtcheva, T., Mantle, P., 2000b. Susceptibility to secondary bacterial infections in growing pigs as an early response in ochratoxicosis. Experimental and Toxicologic Pathology 52, 287–296. Stoev, S.D., Daskalov, H., Radic, B., Domijan, A., Peraica, M., 2002a. Spontaneous mycotoxic nephropathy in Bulgarian chickens with unclarified mycotoxin aetiology. Veterinary Research 33, 83–94. Stoev, S.D., Koynarsky, V., Mantle, P.G., 2002b. Clinicomorphological studies in chicks fed ochratoxin A while simultaneously developing coccidiosis. Veterinary Research Communications 26, 189–204. Stoev, S.D., Djuvinov, D., Mirtcheva, T., Pavlov, D., Mantle, P., 2002c. Studies on some feed additives giving partial protection against ochratoxin A toxicity in chicks. Toxicology Letters 135, 33–50. Stoev, S.D., Stefanov, M., Denev, S., Radic, B., Domijan, A-M., Peraica, M., 2004. Experimental mycotoxicosis in chickens induced by ochratoxin A and penicillic acid and intervention by natural plant extracts. Veterinary Research Communications 28, 727–746. Tapia, M.O., Seawright, A.A., 1984. Experimental ochratoxicosis in pigs. Australian Veterinary Journal 61, 219–222. Williams, R.B., 1973. Effects of different infection rates on the oocyst production of Eimeria acervulina or Eimeria tenella in the chicken. Parasitology 67, 279–288.
Experimental coccidiosis provoked by Eimeria acervulina in chicks simultaneously fed on ochratoxin A contaminated diet V. Koynarski a, S. Stoev
b
b,*
, N. Grozeva b, T. Mirtcheva c, H. Daskalov d, J. Mitev e, P. Mantle f
a Department of Parasitology, Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria Department of General and Clinical pathology, Faculty of Veterinary Medicine, Trakia University, Stara Zagora 6000, Bulgaria c Department of Biochemistry, Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria d Department of Veterinary Hygiene, Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria e Department of Ecology and Zoochygiene, Faculty of Agriculture, Trakia University, Stara Zagora, Bulgaria f Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK
Accepted 5 July 2006
Abstract The progression of coccidiosis provoked by Eimeria acervulina was followed in chicks fed on OTA-contaminated as well as on OTAfree diets. More heavy progress of duodenal coccidiosis, including mortality, occurred in OTA-treated chicks as can be seen from the higher value of lesion (3.50) and oocyst (31.65) indices. A stronger decrease of serum total protein was found in OTA-treated chicks (22.80 g/l) than in chicks infected with E. acervulina(24.20 g/l), but that decrease was strongest in chicks treated with OTA and simultaneously infected with E. acervulina (19.71 g/l). The serum concentration of uric acid was significantly increased in all chicks exposed to OTA, most notably in those additionally infected with E. acervulina (1020.6 (l mol/L), whereas the serum enzyme activity of AST was increased only in chicks infected with E. acervulina and highest in those fed OTA contaminated diet (122.2 U/L). OTA induced degenerative changes in kidneys, liver and heart as well as a depletion of lymphoid tissue in the lymphoid organs and a decrease of body weight. Coccidiosis induced only a slight growth depression and duodenal hemorrhages in addition to characteristic duodenal damages. The impairment of kidney function, histopathological changes and general growth depression were stronger when chicks infected with E. acervulina were also given OTA. 2006 Elsevier Ltd. All rights reserved. Keywords: Ochratoxicosis; Ochratoxin A; Coccidiosis; E. acervulina; Pathology
1. Introduction Ochratoxin A (OTA) is a widespread mycotoxin in lots of components of animal feed. Therefore, the significance of this mycotoxin for animal health has been comprehensively reviewed (Kuiper-Goodman and Scot, 1989; Marquardt and Frolich, 1992). The most important economic problems reported when OTA-contaminated feed was given in chicks were the growth depression and increased mortality, although some other specific damages on many *
Corresponding author. Fax: +359 42 670624. E-mail address: [email protected] (S. Stoev).
0034-5288/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2006.07.004
tissues were also noted (Huff et al., 1974; Dwivedi and Burns, 1984, 1986; Stoev et al., 2000a, 2002b,c, 2004). The increased mortality among chicks or pigs fed on OTA-contaminated diet was seen to be due to OTA-provoked susceptibility to secondary bacterial infections (Stoev et al., 2000b) or to a heavy progression of some often encountered parasitic diseases (Stoev et al., 2002b), because of the suppression in both humoral and cell-mediated immune response in such animals as an aspect of ochratoxicosis (Burns and Dwivedi, 1986; Stoev et al., 2000a,b, 2002c). Our observations on naturally encountered chick nephropathy in Bulgaria have identified a rather high incidence of mortality which can only partly be attributed to
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OTA-contaminated feed (Stoev et al., 2002a). The descriptions of pathology of spontaneous chick nephropathy vary considerably, especially if another secondary disease arises spontaneously along with OTA-induced nephropathy in chicks (Stoev et al., 2002b) that can occur frequently because of the immunosuppression accompanying this nephropathy (Stoev et al., 2000a,b). Therefore, it is important to clarify the complex clinicomorphological findings in such cases, which derive from both diseases as well as to follow the progression of coccidiosis in the same cases. At present, there are very scarce and contradictious data on progression of coccidiosis in chicks fed on OTA-contaminated diet. For example, Huff and Ruff (1982) reported an increase of serum uric acid and an increase of relative weight of liver in chicks fed OTA and infected with E. tenella, which was confirmed by Stoev et al. (2002b), but both the relative weight of liver and the uric acid concentration in serum were decreased in chicks fed OTA and infected with E. acervulina (Huff and Ruff, 1982). That’s why, we decided to extend our recent study on progression of coccidiosis provoked by E. tenella in chicks fed on OTAcontaminated diet (Stoev et al., 2002b) and included E. acervulina in our further investigations in this area.
Table 1 Experimental design Group
OTA in feed between days 1 and 14 (ppm – mg/kg)
Inoculation with E. acervulina – day 7 (4 · 105 oocysts per chick per os)
I II III
None 4 4
E. acervulina None E. acervulina
Control
None
None
Feed levels of ochratoxin A (given between days 1 and 14 of the experiment) and inoculation with E. acervulina (at day 7 of the experiment) of various experimental groups of chicks.
2.3. Measurements The chicks were weighed at the beginning of the experiment and after one and two weeks. For following the pathomorphological changes in various internal organs as well as oocyst and lesion indices the chicks from each group were slaughtered two weeks after the beginning of the experiment or one week after the infection with E. acervulina, as the first mortalities occurred in group III in that time (Table 1).
2. Materials and methods
2.4. Histological examination
2.1. OTA production
Tissues for histological examination were taken from kidneys, liver, lung, heart, thymus, bursa of Fabricius, spleen, intestine, cerebellum, brain and medulla and fixed in 10% neutral buffered formalin. The fixed tissues were processed for paraffin embedding, sectioned at 6 lm and stained with haematoxylin–eosin.
Aspergillus ochraceus (isolate D2306, as used by Tapia and Seawright, 1984; and Stoev et al., 2000b, 2002b) was grown on sterilized shredded wheat (40 g) in 500 ml conical flasks, moistened by a 40% (w/v) addition of sterile water and incubated on a rotary shaker at 27 C for 2 weeks (Harris and Mantle, 2001). The brown granular product, which bore no obvious sign of fungal growth or sporulation, was sterilized at 80 C for 1 h and stored at 20 C. A sample was analyzed for ochratoxins; batches typically contained OTA (about 2 mg/g) and relatively small component of the biologically-inactive alkaloid ochratoxin B (deschloro-ochratoxin A). No other mycotoxins were produced in this solid substrate fermentation process and the necessary dilution by approximately 103 when homogenized into chick ration made only a minimal addition of other components of the molded shredded wheat substrate (Stoev et al., 2000b, 2002b).
2.5. Parasitological examination The required quantity of oocysts was obtained from naturally susceptible chicks (two-weeks of age) inoculated experimentally with oocysts of E. acervulina according to Lozanov (1980). The oocysts were preserved in 2.5% solution of potassium bichromate (K2Cr4O7) at 4 C until used for inoculation (Table 1). The oocyst index in slaughtered chicks was examined according to Cucler (1959), whereas the lesion index in the same chicks was evaluated according to Johnson and Reid (1970) (Table 2). 2.6. Hematological and biochemical examination
2.2. Experimental design Specific pathogen-free chicks (Plymouth Rock) were purchased at a week of age and housed in wire floor cages with continuous infra-red lighting at a temperature suitable for their age. Commercial complete standard feed without any medication (Smesler, Stara Zagora, Bulgaria) with or without added OTA was available ad libitum. The chicks were housed in 3 experimental and 1 control groups (12 birds in each) and treated either or both with OTA and E. acervulina (Table 1).
Blood samples were taken at slaughter at the end of the 14th day of the experiment and examined for various biochemical parameters within 1–2 h of collection, immediately after separation of serum. The serum total protein was measured by Bio-La-Test (Lachema Diagnostica, Brno, Czech Republic) and uric acid by EnzUric-FT-test (Labordiagnostica, Gopecke, Germany). The serum enzyme activity of AST (asparate-aminotransferase) was measured by Cormay test (Smolenskiego, Warsaw, Poland).
V. Koynarski et al. / Research in Veterinary Science 82 (2007) 225–231 Table 2 Mean values of lesion and oocyst indices in slaughtered chicks (7 days after inoculation with E. acervulina). Group
Number of examined chicks
Lesion indexb
Oocyst indexc
I (E.ac.) II (OTA) III (OTA + E.ac.)
12 12 8
2.41 ± 0.31a 0 3.50 ± 0.26
26.31 ± 0.96a 0 31.65 ± 0.52
Control
12
0
0
± SEM (standard error of the mean). a Significant difference compared to group III (p < 0.05). b Lesion index (the maximum is 4) was examined using the following scheme: assessment 1 – scattered white plaque-like lesions confined to the duodenal mucosa (maximum 5 lesions per square centimeter); assessment 2 – lesions are much closer together, but not coalescent; the intestinal wall shows no thickening; assessment 3 – lesions are numerous enough to cause coalescence in the lesion size, giving the duodenal mucosa a coated appearance; the intestinal wall is thickened and the content is watery ; assessment 4 – coalescing of the lesions is much more complete and no distinctive lesions may appear on the duodenal mucosa; the intestinal wall is considerably thickened. c Oocyst index (the maximum is 40) was examined using the following scheme: assessment 0 – from 0 up to 0.1 million oocysts in the intestine for chick; assessment 1 – from 0.1 up to 1 million oocysts; assessment 10 – from 1 up to 5 millions oocysts; assessment 20 – from 5 up to 10 millions oocysts; assessment 40 – more than 10 millions oocysts.
2.7. Statistical methods Non-parametric Mann–Whitney in addition to Student’s t-test was used to estimate significant differences between the mean values of various parameters in different groups of chicks. 3. Results 3.1. Clinical observation Diarrhoea was the main clinical sign seen first in chicks given OTA (groups II and III) on day 3 from the beginning of the experiment, but the same was also seen in chicks of group I, 5 or 6 days after inoculation with E. acervulina and was most pronounced in chicks infected with E. acervulina and simultaneously given OTA (group III) at that time. Other clinical signs, depression, weakness and dullness, ruffled feathers, reduction of feed intake and growth depression occurred in all experimental groups though mainly in groups II and III. One chick from group III dead at
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day 13 and three more dead at day 14 of the experiment as was expected from the higher values of lesion and oocyst indices (Table 2) and the significant decrease of body weight (Table 3) in slaughtered chicks of the same OTAtreated group. 3.2. Body weight A statistically significant decrease of body weight was observed in all experimental groups compared to the control group, but was most marked in groups given OTA. A significant decrease of body weight was seen in chicks given OTA and simultaneously infected with E. acervulina compared to chicks given only OTA (Table 3). 3.3. Biochemical and hematological findings Total protein in serum was significantly decreased in chicks of all experimental groups, most notably in those treated with OTA and especially in group treated with OTA and E. acervulina simultaneously (Table 4). A significant increase was observed in serum uric acid in all OTA-treated groups, but was highest in chicks additionally infected with E. acervulina (Table 4). Serum enzyme activity of AST was somewhat increased in all experimental groups, but that increase was confident only in groups infected with E. acervulina (Table 4). 3.4. Gross pathology and parasitological findings There were a few subcutaneous muscular hemorrhages in chicks of OTA-treated groups II and III. The kidneys and liver in these chicks were congested and enlarged. Their gall bladder was also distended by bile. The mucosal surface of small intestine was hyperemic and covered with mucous fluid. The pancreas was pink-coloured. The meninges were slightly hyperemic. In chicks infected with E. acervulina (groups I and III) there were various white plaque-like lesions confined to the duodenal mucosa, which sometimes coalesced, most notably in OTA-treated chicks, giving the intestinal mucosa a coated appearance. Also, the mucosal surface of duodenum was hyperemic and coated with mucous fluid. In addition, some red spots (2–3 mm diameter) were seen under duodenal serosa. The intestinal wall of the duode-
Table 3 Mean values of body weight (b.w.) in chicks of various experimental groups at the beginning and at end of the first and second weeks of the experiment Group
Day 1 b.w. (g)
Day 7 Number of chicks
b.w. (g) a
Day 14 Number of chicks
b.w. (g) a,*
Number of chicks
I (E.ac.) II (OTA) III (OTA + E.ac.)
85 ± 4.0 83 ± 3.5 86 ± 2.8
12 12 12
152.9 ± 7.7 103.1* ± 5.5 95.4* ± 6.0
12 12 12
243.7 ± 11.3 155.4a,* ± 12.7 110.0* ± 9.5
12 12 8
Control
84 ± 3.6
12
160.4a ± 6.6
12
314.6a ± 16.8
12
± SEM (standard error of the mean). a Significant difference compared to group III (p < 0.05). * Significant difference compared to controls (p < 0.05).
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Table 4 Mean serum values of total protein, AST (asparate-aminotransferase) and uric acid in chicks of various experimental groups at day 14 of the experiment. Group
Total protein (g/l) a,*
AST (U/L) *
Uric acid (lmol/L) a
Number of chicks
I (E.ac.) II (OTA) III (OTA + E.ac.)
24.20 ± 0.89 22.80* ± 1.74 19.71* ± 0.81
114.8 ± 4.7 99.6a ± 4.8 122.2* ± 7.1
165.5 ± 32.6 872.6* ± 96.7 1020.6* ± 212.4
12 12 8
Control
35.40a ± 0.87
79.0a ± 8.6
214.6a ± 20.9
12
± SEM (standard error of the mean). a Significant difference compared to group III (p < 0.05). * Significant difference compared to controls (p < 0.05).
num was often thickened and the content was watery mainly in OTA-treated chicks as can be seen from the lesion indices (Table 2). The number of oocysts in the intestine and the values of the oocyst indices were also clearly higher in chicks given OTA in diet (Table 2). 3.5. Histopathology No histopathological changes were found in internal organs of control chicks. In kidneys of OTA-treated groups (II and III) peritubular capillaries were congested. Degenerative changes (granular degeneration or cloudy swelling as well as karyopycnosis or karyolysis) in epithelial cells of the proximal convoluted tubules were observed (Fig. 1) as well as focal mononuclear cells infiltration was seen in the renal interstice. Some of the tubules contained necrotic debris, granular or hyaline casts in the lumen (Fig. 2). A limited proliferation of connective tissue and activation of capillary endothelium were also noticed in the same chicks. These damages were stronger in chicks given OTA and E. acervulina (group III) than in those given OTA alone (group II). In chicks treated with E. acervulina alone (group I) only a slight hyperemia of peritubular capillaries and scarce granular degeneration in epithelial cells of the proximal convoluted tubules were seen sometimes. Cloudy swelling, granular or vacuolar degeneration were seen in the livers of OTA-treated groups, but the
same were more marked in group III, infected also by E. acervulina(Fig. 3). Also, there was an activation of capillary endothelium and Kupffer’s cells, hyperemia and pericapillary edema, in addition to perivascular infiltration of mononuclear cells. Only a slight hyperemia of capillaries and scarce granular degeneration in hepatocytes were occasionally seen in chicks of group I, treated with E. acervulina alone. Degenerative changes in lymphoid organs were only seen in chicks of groups II and III. There was a pronounced depletion of lymphoid cells in the cortical zone
Fig. 1. Photomicrograph of kidney in chick fed on 4 ppm OTA in diet and infected with E. acervulina. Granular degeneration and karyopycnosis in epithelial cells of the proximal tubules. H/E, scale bar = 33.3 lm.
Fig. 3. Photomicrograph of liver in chick fed on 4 ppm OTA in diet and infected with E. acervulina. Granular and vacuolar degeneration, karyopycnosis and karyolysis in hepatocites H/E, scale bar = 33.3 lm.
Fig. 2. Photomicrograph of kidney in chick fed on 4 ppm OTA in diet. Necrotic debris and hyaline drops in the lumen of some tubules. H/E, scale bar = 33.3 lm.
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of the thymus and sometimes the region between medulla and cortex was not well defined or the cortex became very thin. Similar depletion of lymphoid cells (Fig. 4) and degenerative changes (karyopyknosis and karyorrhexis) in the lymph follicles were seen in the bursa of Fabricius. A slight cellular depletion or degenerative changes were also observed in the white pulp of germinal centres in the spleen of chicks in the same groups. No histopathological changes were seen in the lymphoid organs in chicks of group I, excepting the slight hyperplasia of the white pulp of the spleen. Degenerative and slight necrotic changes as well as a desquamation of surface- or glandular mucosal epithelium of duodenum and jejunum were seen in chicks of groups II (Fig. 5) and III, but the degenerative changes in the duodenum were stronger in the chicks of group III. Small petechial hemorrhages, hyperemia of vessels, slight mononuclear infiltration in the lamina propria, lots of oocysts and third generation of schizonts, in addition to local degenerative changes of surface- or glandular epithelium were also seen in the mucosa of duodenum in chicks
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Fig. 6. Photomicrograph of duodenal mucosa in chick fed on 4 ppm OTA in diet and infected with E. acervulina. Presence of many preliminary forms of oocysts (schizonts) in intestinal mucosa. Damaged distal part of the villi. H/E, scale bar = 50 lm.
Fig. 7. Photomicrograph of brain in chick fed on 4 ppm OTA in diet and infected with E. acervulina.A pericellular and pericapillary edema. H/E, scale bar = 50 lm. Fig. 4. Photomicrograph of bursa of Fabricius in chick fed on 4 ppm OTA in diet and infected with E. acervulina. Depletion of lymphoid cells in the central part of the lymph follicles. H/E, scale bar = 50 lm.
Fig. 5. Photomicrograph of duodenal mucosa in chick fed on 4 ppm OTA in diet. Degenerative changes and desquamation of glandular- and surface mucosal epithelium, including totally damaged villi. H/E, scale bar = 50 lm.
infected with E. acervulina (groups I and III), most notably in group III (Fig. 6). The distal part of the villi of duodenal mucosa was often with desquamated epithelium or totally damaged in the chicks of the same groups, most notably in group III. A perivascular edema, lytic changes and irregular staining, as a result of the increased eosinophilia of some myofibrils were seen in the heart of chicks of groups II and III. Slight granular degeneration of myofibrils and activation of capillary endothelium were also observed in these chicks. In chicks treated with E. acervulina alone (group I) only a hyperemia of vessels, slight perivascular mononuclear infiltration or scarce granular degeneration of myofibrils were rarely seen. In the lung, there was only a perivascular or peribronchial mononuclear cell infiltration and slight edematous changes in the chicks of OTA-treated groups. In the brain, a pericellular or pericapillary edema was seen in chicks of groups II and III (Fig. 7). In neurons and glia cells of the brain in the same chicks there were
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Fig. 8. Photomicrograph of cerebellum in chick fed on 4 ppm OTA in diet and infected with E. acervulina. Edema in the region of the Purkinje’s cells and slightly in the granular layer. Lytic and pycnotic changes in Purkinje’s cells. H/E, scale bar = 50 lm.
slight lytic changes. In the cerebellum of these chicks, edema and slight degenerative changes were observed in the region of the Purkinje’s cells and rarely in molecular or granular layers (Fig. 8). Slight edematous changes in the white substance of the cerebellum were occasionally seen in the chicks of groups II and III. In the lumbosacral region of the medulla, slight lytic changes in neurons were also noticed in some chicks of the same groups. Only a slight hyperemia of capillaries and scarce lytic changes in neurons were occasionally seen in chicks of group I, treated with E. acervulina alone. 4. Discussion It was found that intensity of clinical signs, macroscopic and histopathological changes, the growth depression as well as impairment of kidney function, expressed by serum levels of uric acid, were stronger when chicks infected with E. acervulina were also treated with OTA. Moreover, the coccidiosis progresses in a heavy way and rapidly in OTA-treated chicks, as perceived from the changes in lesion and oocyst indices and especially from the chick mortality. That could be explained by OTA-provoked immunosuppression and following sensitivity to various infectious and parasitic diseases (Stoev et al., 2000a,b, 2002b). The growth depression in OTA-treated chicks, according to some authors (Mohiudin et al., 1993), may be due to the impairment of protein synthesis by OTA. The histopathological changes in OTA-treated chicks, especially those found in kidneys, corresponded well to the deviation of some biochemical parameters, as the increase in the serum concentration of uric acid, which showed impairment in kidney function was evident. The degenerative changes in the epithelial cells of kidneys and liver probably are due to the route of elimination of OTA via kidneys and partly via liver, due to enterohepatic
recirculation and hepatobiliary way of excretion of OTA (Fuchs, 1988), exerting direct toxic effect of OTA on these organs (Dwivedi and Burns, 1984; Stoev et al., 2000a). The slight granular degeneration in hepatocytes in chicks treated with E. acervulina alone could be due to the resorption of toxic products as a consequence of decomposition of necrotic epithelial debris in the intestinal content (Lozanov, 1983). The degenerative changes and the depletion of lymphoid cells in the lymphoid organs in OTA-treated chicks can be in good agreement with immunosuppressive effects that OTA can exerts in contamination levels 2–4 ppm, as reported by some authors (Holmberg et al., 1988; Harvey et al., 1992; Kozaczynski, 1994). Therefore, a heavy progression of some infectious (Stoev et al., 2000a,b) and even parasitic diseases (Stoev et al., 2002b) could be expected as was demonstrated in the present experiment. The OTAprovoked immunosuppression could be explained by the inhibition of protein synthesis and the subsequent delay in cell division in the immune system (Harvey et al., 1992). The impaired protein synthesis in lymphocytes could lead to the impairment in their activation, differentiation and proliferation. In addition, the decreased phagocytic activity of natural killer cells and T-killer cells, which is probably due to a decrease of basal interferon (Harvey et al., 1992), may additionally deteriorate the immune response of OTA-treated chicks. By the present experiment, it was demonstrated in practice, how the known OTA suppression of humoral and cellular immunity (NNT, 1991; Stoev et al., 2000a,b) can make worse the progression of coccidiosis provoked by E. acervulina, and can also lead to mortality among chicks at day 7 of the infection that cannot be commonly provoked by E. acervulina (Williams, 1973; Lozanov, 1983; Holdworth et al., 2004). Diarrhoea and growth depression in chicks were often reported as the main clinical signs in coccidiosis provoked by E. acervulina, but no mortality was seen in such chicks (Lozanov, 1983). The edematous changes seen in various internal organs, in addition to some muscular hemorrhages, might be due to vascular damages, provoked by OTA (Stoev et al., 2000a). Furthermore, a disturbance of blood clotting due to a reduction in the concentration of fibrinogen in the blood and an increase in the prothrombin time, observed as a response of ochratoxicosis (Doerr et al., 1981; Prior and Sisodia, 1978), could also contribute to these muscular hemorrhages. However, the scarce petechial hemorrhages in the duodenum are commonly observed, because of localization of E. acervulina in this place (Johnson and Reid, 1970). It can be supposed that the significant decrease in the serum total protein could be due to the impairment of protein synthesis, provoked by OTA, as has been previously reported (Mohiudin et al., 1993; Stoev et al., 1999, 2000a, 2002b), but the slight decrease in the serum total protein in the chicks infected only with E. acervulina is likely a consequence of progression of coccidiosis and can explain the
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most pronounced decrease in this parameter in the chicks treated with E. acervulina and OTA simultaneously. The strong increase in the serum level of uric acid in both groups treated with OTA showed that the function of kidneys was significantly impaired, which is in agreement with our previous investigations on OTA-toxicity (Stoev et al., 2000a, 2002b). However, the results of the present study contradict with the conclusion made by Huff and Ruff (1982) that the serum concentration of uric acid decreased in chicks fed OTA and simultaneously infected with E. acervulina as was reported in the same paper. Concerning the serum enzyme activity of AST, it can be concluded that the strongest increase in this parameter in the chicks fed OTA and simultaneously infected with E. acervulina may be due to the strongest degenerative changes in the internal organs of the same chicks. Finally, the present experiment was demonstrated in practice that some often encountered parasitic diseases, as coccidiosis provoked by E. acervulina, can progress in a more heavy way in OTA-treated chicks, and can also lead to mortality among chicks that cannot be commonly provoked by E. acervulina. Such mortality was only characteristic for coccidiosis provoked by E. tenella,but even in such cases coccidiosis developed more rapidly and heavily in OTA-treated chicks as was seen from our previous experiment (Stoev et al., 2002b). However, our observation is in contrast with the study made by Huff and Ruff (1982), which found less lesions in chicks challenged with either E. acervulina or E. tenella and fed OTA compared to chicks not fed OTA. The clinicomorphological finding in such cases may be complicated, because it derives from both diseases. Acknowledgement This study was financially supported by the foundation of Ministry of science and education in Bulgaria. References Burns, R.B., Dwivedi, P., 1986. The natural occurrence of ochratoxin A and its effects in poultry. A review. Part 2. Pathology and immunology. World’s Poultry Science Journal 42, 48–55. Cucler, A.C., 1959. The Laboratory Evaluation of Coccidiostatic Drugs. In: Conference on Methods of Testing Coccidiostats. Merc Chemical Division, Rahway, NJ, Sec. 2, 1–14. Doerr, J.A., Huff, W.E., Hamilton, B.P., Lillehoj, E.B., 1981. Severe coagulopathy in young chickens produced by ochratoxin A. Toxicology and Applied Pharmacology 59, 157–163. Dwivedi, P., Burns, R.B., 1984. Pathology of ochratoxicosis A in young broiler chicks. Research in Veterinary Science 36, 92–103. Dwivedi, P., Burns, R.B., 1986. The natural occurrence of ochratoxin A and its effects in poultry. A review. Part 1. Epidemiology and toxicity. World’s Poultry Science Journal 42, 32–47. Fuchs, R., 1988. Distribution and fate of ochratoxin A in experimental animals. Doctoral thesis, Uppsala, pp. 13–41. Harris, J.P., Mantle, P.G., 2001. Biosynthesis of ochratoxins by Aspergillus ochraceus. Phytochemistry 58, 709–716. Harvey, R.B., Elissalde, M.H., Kubena, L., Weaver, E.A., Corrier, D.E., Clement, B.A., 1992. Immunotoxicity of ochratoxin A to growing gilts. American Journal of Veterinary Research 53, 1966–1970.
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